The present invention relates generally to antibiotics and antimicrobial derivatives. More particularly, the present invention relates to intermediates useful for synthesizing laspartomycin derivatives as well as the laspartomycin derivatives.
Laspartomycin (Umezawa et al., U.S. Pat. No. 3,639,582; Naganawa et al., 1968, J. Antibiot., 21, 55; Naganawa et al., 1970, J. Antibiot., 23, 423 which are herein incorporated by reference) is closely related to antibiotics such as zaomycin (Kuroya, 1960, Antibiotics Ann., 194; Kuroya, JP 8150), crystalomycin (Gauze et al., 1957, Antibiotiki, 2, 9), aspartocin (Shay et al., 1960, Antibiotics Annual, 194; Hausman et al., 1964, Antimicrob. Ag. Chemother., 352; Hausman et al., 1969, J. Antibiot., 22, 207; Martin et al., 1960, J. Am. Chem. Soc., 2079), amphomycin (Bodanszky et. al., 1973, J. Am. Chem. Soc., 95, 2352), glumamycin (Fujino et al., 1965, Bull. Chem. Soc. Jap., 38, 515), daptomycin (Debono et. al., 1988, J. Antibiotics, 41, 1093), Antibiotic A-1437 (Hammann et. al., EP 0 629 636 B1; Lattrell et al., U.S. Pat. No. 5,629,288), Antibiotic A54145 (Fukada et al., U.S. Pat. No. 5,039,789; Boeck et al., 1990, J. Antibiotics, 43, 587), and tsushimycin (Shoji et. al., 1968, J. Antibiot., 21, 439). The above compounds are lipopeptide antibiotics which typically inhibit gram positive bacteria. Generally, lipopeptide antibiotics consist of either a cyclic core peptide or a cyclic core depsipeptide acylated with a lipophilic fragment such as an unsaturated fatty acid.
Laspartomycin, produced by fermenting the microorganism Streptomyces viridochromogenes var. komabensis, was first isolated while screening for compounds active against resistant staphylococci (Naganawa et al., 1968, J. Antibiot., 21, 55; Umezawa et al., U.S. Pat. No. 3,639,582). Laspartomycin was characterized by conventional methods and was shown to be active against a variety of gram positive bacteria, including staphylococci and some fungi (id.). Elemental analysis and amino acid analysis provided a molecular weight of about 1827 for the lipopeptide antibiotic, while amino acid analysis indicated the presence of the amino acids threonine and diaminobutryic acid in the peptide portion of laspartomycin (id.).
In other studies, the major lipophilic fragment of laspartomycin was shown to be trans-2-isopentadecanoic acid 2, illustrated below (Naganawa et al., 1970, J. Antibiot. 23, 423). In contrast, the lipophilic portions of antibiotics such as aspartocin (Hausmann et al., 1963, Antimicr. Agents and Chemoth., 352, 1962), glumamycin (Inoue, 1962, Bull. Chem. Soc. Jap., 35, 1255), tsushimycin (Shoji et al., 1968, J. Antibiot., 21, 439) and amphomycin (Shoji et al., 1969, J. Antibiot., 22, 473) are all derived from cis xcex2-xcex3 unsaturated carboxylic acids. 
The results described in the instant Application indicate that the amino acid analysis and the molecular weight disclosed in the art are incorrect (Umezawa et al., U.S. Pat. No. 3,639,582; Naganawa et al., 1968, J. Antibiot., 21, 55). In particular current studies disclosed in this Application show that the peptide core of laspartomycin contains novel amino acids not found in other known lipopeptide antibacterial antibiotics. For example, laspartomycin is the only member of the antibacterial lipopeptide family that contains diaminopropionic acid in the peptide core. Amphomycin, aspartocin, zaomycin, tsushimycin, and antibiotic A-1437 contain, instead, 2,3-diaminobutyric acid in the peptide portion of the molecule (Kuroya, 1960, Antibiotics Ann., 194; Gauze et al.,1957, Antibiotiki, 2, 9; Shay et al., 1960, Antibiotics Annual, 194-198; Hausman et al., 1964, Antimicrob. Ag. Chemother., 352; Hausman et al., 1969, J. Antibiot., 22, 207; Martin et al., 1960, J. Am. Chem. Soc., 2079; Bodanszky et. al., 1973, J. Am. Chem. Soc., 95, 2352; Fujino et al., 1965, Bull. Chem. Soc. Jap., 38, 515; Hammann et. al., EP 0 629 636 B1; Lattrell et al., U.S. Pat. No. 5,629,288; Shoji et al., 1968, J. Antibiot., 21, 439). Additionally, laspartomycin contains allothreonine, which is not found in the other known lipopeptides. Further laspartomycin is the smallest of the known lipopeptides with a molecular weight of about 1247 for the cyclic core peptide acylated with compound 2.
Despite the efficacy of laspartomycin against gram positive bacteria, the medicinal chemistry of this lipopeptide antibacterial antibiotic has remained largely unexplored. However, given the recent dramatic rise of antibiotic-resistant pathogens and infectious diseases, caused in part, by frequent over use of antibiotics, the need for new antimicrobial agents is urgent (Cohen et al., 1992, Science, 257, 1050-1055). Specifically, methicillin resistant bacteria are a particular problem since they are also resistant to a wide variety of antibiotics other than methicillin (Yoshida et al., U.S. Pat. No. 5,171,836). Gram positive bacteria, such as Staphylococci, which cause persistent infections, are especially dangerous when methicillin resistant. Even more alarmingly, strains of Enterococcus faecium that are resistant to vancomycin have been recently observed (Moellering, 1990, Clin. Microbiol. Rev., 3, 46). Strains resistant to vancomycin pose a serious health threat to society since vancomycin is the antibiotic of last resort for several harmful pathogens. Thus, there is a general need for antibiotic agents and a specific need for antibiotic agents that are active against microbes resistant to methicillin or vancomycin.
The present invention addresses this and other needs in the art by providing antimicrobial laspartomycin derivatives, pharmaceutical compositions of antimicrobial laspartomycin derivatives, methods for making antimicrobial laspartomycin derivatives, methods for inhibiting microbial growth and methods for treating or preventing microbial infections in a subject. The present invention also provides a laspartomycin core peptide, methods for making the laspartomycin core peptide and a laspartomycin core peptide derivative and methods for making the laspartomycin core peptide derivative all of which are all useful in synthesizing antimicrobial laspartomycin derivatives.
In one aspect, the present invention provides a laspartomycin core peptide derivative that may be used as a key intermediate in the synthesis of antimicrobial laspartomycin derivatives. An essential part of the laspartomycin core peptide derivative is a core cyclic peptide attached to a nitrogen atom which may be part of a variety of functional groups such as, for example, a carbamate, amide or sulfonamide.
In one embodiment, the laspartomycin core peptide derivative includes a linker which is typically attached to the nitrogen of the core cyclic peptide. The linker may be derived from compounds such as amino acids, polyamides, polyamines, polyethers, polysulfonamides or other linkers known to those of skill in the art. The linker typically includes a linking group which may be any chemical functionality that can participate in covalent bond formation. The linking group provides a site for further modification of the laspartomycin core peptide derivative. For example, the linking group may be modified with a lipophilic moiety to provide a laspartomycin derivative of the invention.
Thus, in one illustrative embodiment, the present invention provides a laspartomycin core peptide derivative according to structural formula (I):
Y1xe2x80x94Lxe2x80x94X1xe2x80x94N(R1)xe2x80x94Rxe2x80x83xe2x80x83(I)
or a salt or hydrate thereof, wherein either:
(i) Y1xe2x80x94Lxe2x80x94X1 taken together is hydrogen; or
(ii) Y1 is a linking group;
L is a linker;
X1 is selected from the group consisting of xe2x80x94COxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94CSxe2x80x94, xe2x80x94POxe2x80x94, xe2x80x94OPOxe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94NHCOxe2x80x94 and xe2x80x94NR1COxe2x80x94;
N is nitrogen;
R1 is selected from the group consisting of hydrogen, (C1-C10) alkyl optionally substituted with one or more of the same or different R2 groups, (C1-C10) heteroalkyl optionally substituted with one or more of the same or different R2 groups, (C5-C10) aryl optionally substituted with one or more of the same or different R2 groups, (C5-C15) arylaryl optionally substituted with one or more of the same or different R2 groups, (C5-C15) biaryl optionally substituted with one or more of the same or different R2 groups, five to ten membered heteroaryl optionally substituted with one or more of the same or different R2 groups, (C6-C16) arylalkyl optionally substituted with one or more of the same or different R2 groups and six to sixteen membered heteroarylalkyl optionally substituted with one or more of the same or different R2 groups;
each R2 is independently selected from the group consisting of xe2x80x94OR3, xe2x80x94SR3, xe2x80x94NR3R3, xe2x80x94CN, xe2x80x94NO2, xe2x80x94N3, xe2x80x94C(O)OR3, xe2x80x94C(O)NR3R3, xe2x80x94C(S)NR3R3, xe2x80x94C(NR3)NR3R3, xe2x80x94CHO, xe2x80x94R3CO, xe2x80x94SO2R3, xe2x80x94SOR3, xe2x80x94PO(OR3)2, xe2x80x94PO(OR3), xe2x80x94CO2H, xe2x80x94SO3H, xe2x80x94PO3H, halogen and trihalomethyl;
each R3 is independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C5-C10) aryl, 5-10 membered heteroaryl, (C6-C16) arylalkyl and six to sixteen membered heteroarylalkyl; and
R is the core cyclic peptide of laspartomycin.
In another aspect, the present invention provides antimicrobial laspartomycin derivatives. The antimicrobial laspartomycin derivatives are generally laspartomycin core peptide derivatives of the invention that have been further modified with a lipophilic moiety. The lipophilic moiety will usually be attached to a linking group covalently bonded to the nitrogen atom of the core peptide derivative.
Thus, in another illustrative embodiment, the present invention provides an antimicrobial laspartomycin derivative according to structural formula (II):
Y2xe2x80x94(X2xe2x80x94X3)xe2x80x94(L)xe2x80x94(X1)xe2x80x94N(R1)xe2x80x94Rxe2x80x83xe2x80x83(II)
or a pharmaceutically acceptable salt or hydrate thereof, wherein:
Y2 is a lipophilic group;
X1 is selected from the group consisting of xe2x80x94COxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94CSxe2x80x94, xe2x80x94POxe2x80x94, xe2x80x94OPOxe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94NHCOxe2x80x94 and xe2x80x94NR1COxe2x80x94;
X2 is a linked group;
X3 is a linked group; and
N, L, R1 and R are as previously defined for Formula (I).
In a third aspect, the present invention provides a method for making a laspartomycin core peptide that includes culturing the microorganism Streptomyces viridochromogenes, ssp. komabensis (ATCC 29814) in a culture medium to provide laspartomycin. Isolation of laspartomycin followed by cleavage of a lipophilic fragment provides the laspartomycin core peptide.
In a fourth aspect, the present invention provides methods for synthesizing a laspartomycin core peptide derivative. A linking moiety may be covalently attached to a laspartomycin core peptide to provide a laspartomycin core peptide derivative.
In a fifth aspect, the present invention provides approaches for synthesizing antimicrobial laspartomycin derivatives. In a first method, a linking moiety may be covalently attached to a laspartomycin core peptide to yield a laspartomycin core peptide derivative. Then, a lipophilic group may be covalently attached to the laspartomycin core peptide derivative to provide an antimicrobial laspartomycin derivative. In a second method, a linking moiety may be covalently attached to a lipophilic group to yield a linker-lipophilic group. Then the linker-lipophilic group may be covalently attached to the laspartomycin core peptide to provide an antimicrobial laspartomycin derivative.
In a sixth aspect, the present invention provides pharmaceutical compositions comprising the antimicrobial laspartomycin derivatives of the invention. The pharmaceutical compositions generally comprise one or more antimicrobial laspartomycin derivatives of the invention, and/or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier, excipient or diluent. The choice of carrier, excipient or diluent will depend upon, among other factors, the desired mode of administration.
In a seventh aspect, the present invention provides methods of inhibiting the growth of microbes such as gram positive bacteria, particularly, methicillin resistant Staphylococcus aureus and vancomycin resistant enterococci. The method generally involves contacting a microbe with one or more antimicrobial laspartomycin derivatives of the invention (or a pharmaceutically-acceptable salt thereof) in an amount effective to inhibit the growth of the microbe. The method may be practical to achieve a bacteriostatic effect, where the growth of the microbe is inhibited, or to achieve a bactericidal effect, where the microbe is killed.
In a final aspect, the present invention provides methods for treating and/or preventing microbial infections in a subject such as human, plant or animal. The methods generally involve administering to a subject one or more of the antimicrobial laspartomycin derivatives or pharmaceutical compositions of the invention in an amount effective to treat or prevent a microbial infection in the human, animal or plant. The antimicrobial laspartomycin derivatives or pharmaceutical compositions may be administered systemically or applied topically, depending on the nature of the microbial infection.
As used herein, the following terms are intended to have the following meanings.
xe2x80x9cLaspartomycin:xe2x80x9d refers to a mixture of at least three different compounds produced by culturing the microorganism Streptomyces viridochromogenes, ssp. komabensis (ATCC 29814) in a culture medium. It should be understood that the structure of the lipophilic side chain is different in the three compounds. 
The major component of laspartomycin (typically around 80% under the fermentation and processing conditions used in this Application) is acylated with the C-15 xcex1-xcex2 unsaturated carboxylic acid 2 shown above to provide C-15 laspartomycin 4 shown below. 
The two minor components are the C-14 and C-16 analogues of the C-15 xcex1-xcex2 unsaturated carboxylic acid 2 shown above. The formulation of the culture medium and the ratio of the medium constituents has a direct effect on the ratio of the components of laspartomycin. Thus, no particular component composition is intended by the use of the term xe2x80x9claspartomycin.xe2x80x9d
xe2x80x9cLipophilic fragment:xe2x80x9d refers to any lipophilic moiety attached to the laspartomycin core peptide that is produced by culturing the microorganism Streptomyces viridochromogenes, ssp. komabensis (ATCC 29814) in a culture medium. Thus, lipophilic fragments include but are not limited to, the C-14, C-15 and C-16 acyl analogues of the C-14, C-15 and C-16 xcex1-xcex2 unsaturated carboxylic acids described above.
xe2x80x9cCore cyclic peptide:xe2x80x9d refers to the cyclic peptide portion of laspartomycin R shown below: 
The dashed line indicates the carbon atom which is bonded to nitrogen in Formulas (I), (II) and (III).
xe2x80x9cLaspartomycin core peptide:xe2x80x9d refers to the peptide portion of laspartomycin after cleavage of at least the lipophilic fragment. The laspartomycin core peptide may be represented by Formula (III) shown below:
RxNHRxe2x80x83xe2x80x83(III)
where Rx is either H or NH2CH(CH2CO2H)COxe2x80x94 and R is the core cyclic peptide of laspartomycin as defined above.
xe2x80x9cAlkylxe2x80x9d refers to a saturated or unsaturated, branched, straight-chain or cyclic monovalent hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne. Typical alkyl groups include, but are not limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.
The term xe2x80x9calkylxe2x80x9d is specifically intended to include groups having any degree or level of saturation, i.e., groups having exclusively single carbon-carbon bonds, groups having one or more double carbon-carbon bonds, groups having one or more triple carbon-carbon bonds and groups having mixtures of single, double and triple carbon-carbon bonds. Where a specific level of saturation is intended, the expressions xe2x80x9calkanyl,xe2x80x9d xe2x80x9calkenyl,xe2x80x9d and xe2x80x9calkynylxe2x80x9d are used. The expression xe2x80x9clower alkylxe2x80x9d refers to alkyl groups comprising from 1 to 8 carbon atoms.
xe2x80x9cAlkanylxe2x80x9d refers to a saturated branched, straight-chain or cyclic alkyl group. Typical alkanyl groups include, but are not limited to, methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, etc.; butyanyls such as butan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like.
xe2x80x9cAlkenylxe2x80x9d refers to an unsaturated branched, straight-chain or cyclic alkyl group having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene. The group may be in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like.
xe2x80x9cAlkynylxe2x80x9d refers to an unsaturated branched, straight-chain or cyclic alkyl group having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne. Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.
xe2x80x9cArylxe2x80x9d refers to a monovalent aromatic hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and the like. In preferred embodiments, the aryl group is (C5-C14) aryl, with (C5-C10) being even more preferred. Particularly preferred aryls are cyclopentadienyl, phenyl and naphthyl.
xe2x80x9cArylaryl:xe2x80x9d refers to a monovalent hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a ring system in which two or more identical or non-identical parent aromatic ring systems are joined directly together by a single bond, where the number of such direct ring junctions is one less than the number of parent aromatic ring systems involved. Typical arylaryl groups include, but are not limited to, biphenyl, triphenyl, phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, and the like. Where the number of carbon atoms in an arylaryl group are specified, the numbers refer to the carbon atoms comprising each parent aromatic ring. For example, (C5-C14) arylaryl is an arylaryl group in which each aromatic ring comprises from 5 to 14 carbons, e.g., biphenyl, triphenyl, binaphthyl, phenylnaphthyl, etc. Preferably, each parent aromatic ring system of an arylaryl group is independently a (C5-C14) aromatic, more preferably a (C5-C10) aromatic. Also preferred are arylaryl groups in which all of the parent aromatic ring systems are identical, e.g., biphenyl, triphenyl, binaphthyl, trinaphthyl, etc.
xe2x80x9cBiaryl:xe2x80x9d refers to an arylaryl group having two identical parent aromatic systems joined directly together by a single bond. Typical biaryl groups include, but are not limited to, biphenyl, binaphthyl, bianthracyl, and the like. Preferably, the aromatic ring systems are (C5-C14) aromatic rings, more preferably (C5-C10) aromatic rings. A particularly preferred biaryl group is biphenyl.
xe2x80x9cArylalkylxe2x80x9d refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like. Where specific alkyl moieties are intended, the nomenclature arylalkanyl, arylakenyl and/or arylalkynyl is used. In preferred embodiments, the arylalkyl group is (C6-C20) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C6) and the aryl moiety is (C5-C14). In particularly preferred embodiments the arylalkyl group is (C6-C13), e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C3) and the aryl moiety is (C5-C10).
xe2x80x9cHeteroarylxe2x80x9d refers to a monovalent heteroaromatic group derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring system. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, arsindole, carbazole, xcex2-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuiran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like. In preferred embodiments, the heteroaryl group is a 5-14 membered heteroaryl, with 5-10 membered heteroaryl being particularly preferred. The most preferred heteroaryl groups are those derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine.
xe2x80x9cHeteroarylalkylxe2x80x9d refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with a heteroaryl group. Where specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylakenyl and/or heterorylalkynyl is used. In preferred embodiments, the heteroarylalkyl group is a 6-20 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-6 membered and the heteroaryl moiety is a 5-14-membered heteroaryl. In particularly preferred embodiments, the heteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety is 1-3 membered and the heteroaryl moiety is a 5-10 membered heteroaryl.
xe2x80x9cSubstituted:xe2x80x9d refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s). Typical substituents include, but are not limited to, xe2x80x94X, xe2x80x94R6, xe2x80x94Oxe2x88x92, xe2x95x90O, xe2x80x94OR, xe2x80x94SR6, xe2x80x94Sxe2x88x92, xe2x95x90S, xe2x80x94NR6R6, xe2x95x90NR6, xe2x80x94CX3, xe2x80x94CF3, xe2x80x94CN, xe2x80x94OCN, xe2x80x94SCN, xe2x80x94NO, xe2x80x94NO2, xe2x95x90N2, xe2x80x94N3, xe2x80x94S(O)2Oxe2x88x92, xe2x80x94S(O)2OH, xe2x80x94S(O)2R6, xe2x80x94OS(O2)Oxe2x88x92, xe2x80x94OS(O)2OH, xe2x80x94OS(O)2R6, xe2x80x94P(O)(Oxe2x88x92)2, xe2x80x94P(O)(OH)(Oxe2x88x92), xe2x80x94OP(O)2(Oxe2x88x92), xe2x80x94C(O)R6, xe2x80x94C(S)R6, xe2x80x94C(O)OR6, xe2x80x94C(O)Oxe2x88x92, xe2x80x94C(S)OR6, and xe2x80x94C(NR6)NR6R6, where each X is independently a halogen; each R6 is independently hydrogen, halogen, alkyl, aryl, arylalkyl, arylaryl, arylheteroalkyl, heteroaryl, heteroarylalkyl xe2x80x94NR7R7, xe2x80x94C(O)R7 or xe2x80x94S(O)2R7; and each R7 is independently hydrogen, alkyl, alkanyl, alkynyl, aryl, arylalkyl, arylheteralkyl, arylaryl, heteroaryl or heteroarylalkyl.
Reference will now be made in detail to preferred embodiments of the invention. While the invention will be described in conjunction with preferred embodiments, it should be understood that it is not intended to limit the invention to this preferred embodiment. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.
The present invention provides a laspartomycin core peptide, laspartomycin core peptide derivatives, antimicrobial laspartomycin derivatives, methods for making the laspartomycin core peptide, methods for making laspartomycin core peptide derivatives, methods for making antimicrobial laspartomycin derivatives, pharmaceutical compositions of antimicrobial laspartomycin derivatives, methods of inhibiting microbial growth and methods for treating and/or preventing microbial infections in a subject.
Those of skill in the art will appreciate that many of the compounds encompassed by generic formulae (I-III) as well as the compound species specifically described herein, may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism and/or stereo isomerism. As the formula drawings within the specification and claims can represent only one of the possible tautomeric, conformational isomeric, enantiomeric or geometric isomeric forms, it should be understood that the invention encompasses any tautomeric, conformational isomeric, enantiomeric and/or geometric isomeric forms of the compounds having one or more of the utilities described herein, as well as mixtures of these various different forms.
4.2.1 xe2x80x94Laspartomycin Core Peptide Derivatives
Laspartomycin core peptide derivatives provide synthetic access to a wide variety of antimicrobial laspartomycin derivatives that may possess greater activity against resistant species than previously described antibiotic agents. The ease with which a wide variety of isolated antimicrobial laspartomycin derivatives may be synthesized from laspartomycin core peptide derivatives may establish a structure-activity relationship for the lipophilic group and/or the linker and linking group. Thus, access to laspartomycin core peptide derivatives may allow for facile investigation of the medicinal chemistry of antimicrobial laspartomycin derivatives.
Laspartomycin core peptide derivatives include compounds described by structural Formula (I):
Y1xe2x80x94Lxe2x80x94X1xe2x80x94N(R1)xe2x80x94Rxe2x80x83xe2x80x83(I)
or a salt or hydrate thereof, wherein either:
(i) Y1xe2x80x94Lxe2x80x94X1 taken together is hydrogen; or
(ii) Y1 is a linking group;
L is a linker;
X1 is selected from the group consisting of xe2x80x94COxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94CSxe2x80x94, xe2x80x94POxe2x80x94, xe2x80x94OPOxe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94NHCOxe2x80x94 and xe2x80x94NR1COxe2x80x94;
N is nitrogen;
R1 is selected from the group consisting of hydrogen, (C1-C10) alkyl optionally substituted with one or more of the same or different R2 groups, (C1-C10) heteroalkyl optionally substituted with one or more of the same or different R2 groups, (C5-C10) aryl optionally substituted with one or more of the same or different R2 groups, (C5-C15) arylaryl optionally substituted with one or more of the same or different R2 groups, (C5-C15) biaryl optionally substituted with one or more of the same or different R2 groups, five to ten membered heteroaryl optionally substituted with one or more of the same or different R2 groups, (C6-C16) arylalkyl optionally substituted with one or more of the same or different R2 groups and six to sixteen membered heteroarylalkyl optionally substituted with one or more of the same or different R2 groups;
each R2 is independently selected from the group consisting of xe2x80x94OR3, xe2x80x94SR3, xe2x80x94NR3R3, xe2x80x94CN, xe2x80x94NO2, xe2x80x94N3, xe2x80x94C(O)OR3, xe2x80x94C(O)NR3R3, xe2x80x94C(S)NR3R3, xe2x80x94C(NR3)NR3R3, xe2x80x94CHO, R3COxe2x80x94, xe2x80x94SO2R3, xe2x80x94SOR3, xe2x80x94PO(OR3)2, xe2x80x94PO(OR3), xe2x80x94CO2H, xe2x80x94SO3H, xe2x80x94PO3H, halogen and trihalomethyl;
each R3 is independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C5-C10) aryl, 5-10 membered heteroaryl, (C6-C16) arylalkyl and six to sixteen membered heteroarylalkyl; and
R is the core cyclic peptide of laspartomycin.
Those of skill in the art will appreciate that the compounds of Formula (I) possess the core cyclic peptide of laspartomycin 5 shown below as a common structural motif. 
Although the core cyclic peptide R is illustrated as comprised of certain amino acids arranged with a particular connectivity, the specific structure depicted is not intended to be limiting. Thus, it will be understood that the illustrated structure is intended merely as a convenient method for representing the actual compound and to the extent it may be found at a later date that this structural representation of the core cyclic peptide of laspartomycin is incorrect, it is not intended to be limiting in any way.
The moiety covalently bonded to the dashed line of structure 5 which represents the core cyclic peptide R in generic formula I is N(R1). Here, N represents nitrogen that is directly attached to the core cyclic peptide R and R1 is a nitrogen substituent.
In a preferred embodiment, R1 is selected from the group consisting of hydrogen, (C1-C10) alkyl optionally substituted with one or more of the same or different R2 groups, (C1-C10) heteroalkyl optionally substituted with one or more of the same or different R2 groups, (C5-C10) aryl optionally substituted with one or more of the same or different R2 groups, (C5-C15) biaryl optionally substituted with one or more of the same or different R2 groups, five to ten membered heteroaryl optionally substituted with one or more of the same or different R2 groups, (C6-C16) arylalkyl optionally substituted with one or more of the same or different R2 groups and six to sixteen membered heteroarylalkyl optionally substituted with one or more of the same or different R2 groups where R2 is a substituent as defined above in Formula (I). In another preferred embodiment, R1 is selected from the group consisting of hydrogen, (C1-C6) alkyl optionally substituted with one or more of the same or different R2 groups, (C3-C7) alkenyl optionally substituted with one or more of the same or different R2 groups, C6 aryl optionally substituted with one or more of the same or different R2 groups, C12 biaryl optionally substituted with one or more of the same or different R2 groups, (C6-C10) arylalkyl optionally substituted with one or more of the same or different R2 groups and (C6-C10) heteroarylalkyl optionally substituted with one or more of the same or different R2 groups. In yet another preferred embodiment, R1 is selected from the group consisting of hydrogen, methyl, allyl, homoallyl, phenyl, substituted phenyl, benzyl and substituted benzyl. In one particularly preferred embodiment, R1 is hydrogen.
Laspartomycin core peptide derivatives may be Hxe2x80x94N(R1)xe2x80x94R when Y1xe2x80x94Lxe2x80x94X1 taken together are hydrogen. In a preferred embodiment, a laspartomycin core peptide derivative is H2Nxe2x80x94R when R1 is hydrogen. Those of skill in the art will appreciate that in this situation the laspartomycin core peptide derivative may be represented by the structural formula 6 shown below, which is identical to the laspartomycin core peptide produced by deacylation of laspartomycin with Actinoplanes utahensis (NRRL 12052), supra. 
In an alternative embodiment, laspartomycin core peptide derivatives can also be described by the formula Y1xe2x80x94Lxe2x80x94X1xe2x80x94N(R1)xe2x80x94R. Generally, X1 may be any kind of chemical functionality that can form a covalent bond with nitrogen known to those of skill in the art. In a exemplary embodiment, X1 is selected from the group consisting of xe2x80x94COxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94CSxe2x80x94, xe2x80x94POxe2x80x94, xe2x80x94OPOxe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94NR1COxe2x80x94. In another preferable embodiment, X1 is xe2x80x94COxe2x80x94 or xe2x80x94SO2xe2x80x94. In a particularly preferred embodiment, X1 is xe2x80x94COxe2x80x94.
Connected to X1 in laspartomycin core peptide derivatives of the form Y1xe2x80x94Lxe2x80x94X1xe2x80x94N(R1)xe2x80x94R is a linking moiety of the formula Y1xe2x80x94L, where L is a linker and Y1 is a linking group. The nature of linker L and linking group Y1 may vary extensively. The linker L may be hydrophilic or hydrophobic, long or short, rigid, semirigid or flexible.
A wide variety of linkers L comprised of stable bonds suitable for spacing linking groups such as Y1 from the core cyclic peptide are known in the art, and include by way of example and not limitation, alkyl, heteroalkyl, acyclic heteroatomic bridges, aryl, arylaryl, arylalkyl, heteroaryl, heteroaryl-heteroaryl, substituted heteroaryl-heteroaryl, heteroarylalkyl, heteroaryl-heteroalkyl and the like. Thus, linker L may include single, double, triple or aromatic carbon-carbon bonds, nitrogen-nitrogen bonds, carbon-nitrogen, carbon-oxygen bonds and/or carbon-sulfur bonds, and may therefor include functionalities such as carbonyls, ethers, thioethers, carboxamides, sulfonamides, ureas, urethanes, hydrazines, etc.
Choosing a suitable linker is within the capabilities of those having skill in the art. For example, where a rigid linker is desired, L may be a rigid polyunsaturated alkyl or an aryl, biaryl, heteroaryl etc. Where a flexible linker is desired, L may be a flexible peptide such as Gly-Gly-Gly or a flexible saturated alkanyl or heteroalkanyl. Hydrophilic linkers may be, for example, polyalcohols or polyethers such as polyalkyleneglycols. Hydrophobic linkers may be, for example, alkyls or aryls.
Preferably, linking group Y1 is capable of mediating formation of a covalent bond with complementary reactive functionality of a lipophilic group to provide an isolated antimicrobial laspartomycin derivative. Accordingly, linking group Y1 may be any reactive functional group known to those of skill in the art. Y1 may be for example, a photochemically activated group, an electrochemically activated group, a free radical donor, a free radical acceptor, a nucleophilic group or an electrophilic group. However, those of skill in the art will recognize that a variety of functional groups which are typically unreactive under certain reaction conditions can be activated to become reactive. Groups that can be activated to become reactive include, e.g., alcohols, carboxylic acids and esters, including salts thereof.
Thus, in a preferred embodiment, Y1 is selected from the group consisting of xe2x80x94NHR1, xe2x80x94NH2, xe2x80x94OH, xe2x80x94SH, xe2x80x94PH, halogen, xe2x80x94CHO, xe2x80x94R1CO, xe2x80x94SO2H, xe2x80x94PO2H, xe2x80x94N3, xe2x80x94CN, xe2x80x94CO2H, xe2x80x94SO3H, xe2x80x94PO3H, xe2x80x94PO2(OR1)H, xe2x80x94CO2R1, xe2x80x94SO3R1 and xe2x80x94PO(OR1)2. In another preferred embodiment, Y1 is selected from the group consisting of xe2x80x94NHR1, xe2x80x94NH2, xe2x80x94OH, xe2x80x94SH, xe2x80x94CHO, xe2x80x94CO2H, R1COxe2x80x94 and xe2x80x94CO2R1. In a particularly preferred embodiment, Y1 is selected from the group consisting of xe2x80x94SH, xe2x80x94NH2, xe2x80x94OH, xe2x80x94CO2H, and xe2x80x94CO2R1.
Some embodiments of Y1xe2x80x94L include for example, compounds where L is xe2x80x94(CH2)nxe2x80x94, n is an integer between 1 and 8, Y1 is selected from the group consisting of xe2x80x94NH2, xe2x80x94OH, xe2x80x94CO2H, and xe2x80x94CO2R1 and the corresponding analogues where any suitable hydrogen is substituted. Other embodiments of Y1xe2x80x94L include any amino acid, which may be for example, a D or L xcex1-amino acid, a xcex2-amino acid or a xcex3-amino acid. Thus, Y1xe2x80x94L may be a dipeptide, a tripeptide or a tetrapeptide comprised of any combination of amino acids (preferably xcex1-amino acids). The polarity of the peptide bond in these peptides may be either Cxe2x86x92N or Nxe2x86x92C.
In a preferred embodiment of the laspartomycin core peptide derivative, R1 is hydrogen, Y1 is selected from the group consisting H2Nxe2x80x94, xe2x80x94OH, xe2x80x94SH, xe2x80x94CO2H, xe2x80x94CO2R, X1 is xe2x80x94COxe2x80x94 and L is selected from the group consisting of: 
or a salt or hydrate thereof, wherein:
n is 0, 1, 2 or 3;
each S1 is selected from the group consisting of hydrogen, (C1-C10) alkyl optionally substituted with one or more of the same or different R4 groups, (C1-C10) heteroalkyl optionally substituted with one or more of the same or different R4 groups, (C5-C10) aryl optionally substituted with one or more of the same or different R4 groups, (C5-C15) arylaryl optionally substituted with one or more of the same or different R4 groups, (C5-C15) biaryl optionally substituted with one or more of the same or different R4 groups, five to ten membered heteroaryl optionally substituted with one or more of the same or different R4 groups, (C6-C16) arylalkyl optionally substituted with one or more of the same or different R4 groups and six to sixteen membered heteroarylalkyl optionally substituted with one or more of the same or different R4 groups;
each R4 is independently selected from the group consisting of xe2x80x94OR5, xe2x80x94SR5, xe2x80x94NR5R5, xe2x80x94CN, xe2x80x94NO2, xe2x80x94N3, xe2x80x94C(O)OR5, xe2x80x94C(O)NR5R5, xe2x80x94C(S)NR5R5, xe2x80x94C(NR5)NR5R5, xe2x80x94CHO, xe2x80x94R5CO, xe2x80x94SO2R5, xe2x80x94SOR5, xe2x80x94PO(OR5)2, xe2x80x94PO(OR5), xe2x80x94CO2H, xe2x80x94SO3H, xe2x80x94PO3H, halogen and trihalomethyl;
each R5 is independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C5-C10) aryl, 5-10 membered heteroaryl, (C6-C16) arylalkyl and six to sixteen membered heteroarylalkyl; and
each K is independently selected from the group consisting of oxygen, nitrogen, sulfur and phosphorus.
In a preferred embodiment, S1 is a side chain of a genetically encoded a amino acid. Exemplary preferred embodiments of Y1xe2x80x94Lxe2x80x94X1xe2x80x94NHxe2x80x94R where K is independently selected from the group consisting of oxygen, nitrogen and sulfur include the following compounds: 
Preferably, in the above illustrated embodiments, Y1 is selected from the group consisting of xe2x80x94SH, xe2x80x94NH2 or xe2x80x94OH. More preferably Y1 is xe2x80x94OH.
In another preferred embodiment of the laspartomycin core peptide derivative, R1 is hydrogen, Y1 is H2Nxe2x80x94, X1 is xe2x80x94COxe2x80x94, n is as previously defined, each S1 is independently as previously defined and L is L1 as previously defined. Preferably, in this embodiment, each S1 is independently a side-chain of a genetically encoded xcex1-amino acid. More preferably, each S1 is independently a side-chain of glycine, asparagine, aspartic acid, glutamine, glutamic acid, tryptophan, phenylalanine, tyrosine, leucine, alanine, isoluecine and valine. Exemplary preferred embodiments of Y1xe2x80x94Lxe2x80x94X1xe2x80x94N(H)xe2x80x94R where each S1 is independently a side-chain of glycine, asparagine, aspartic acid, glutamine, glutamic acid and tryptophan include the following compounds where R and Y1 are as previously defined: 
Preferably, Y1 is selected from the group consisting of xe2x80x94OH, xe2x80x94SH and xe2x80x94NHR1 and xe2x80x94NH2. Most preferably, Y1 is xe2x80x94NH2 and the a amino acids illustrated have the L stereochemistry.
4.2.2 Methods of Making the Laspartomycin Core Peptide
The present invention provides methods for making a laspartomycin core peptide that includes culturing the microorganism Streptomyces viridochromogenes, ssp. komabensis (ATCC 29814) in a culture medium to provide laspartomycin. Isolation of laspartomycin followed by cleavage of a lipophilic fragment provides the laspartomycin core peptide.
Parent cultures of Streptomyces viridochromogenes, ssp. komabensis (ATCC 29814) especially suitable for biochemical synthesis of laspartomycin may be selected by conventional methods known to those of skill in the art. A preferred method for selecting a parent culture which provides improved yields of laspartomycin is described in Example 1.
Growing inocula and inoculating culturing medium are also well known to those of skill in the art and exemplary methods for Streptomyces viridochromogenes, ssp. komabensis are described in Umezawa et al., U.S. Pat. No. 3,639,582, which is herein incorporated by reference, and Example 2.
Generally, any culturing medium which supports Streptomyces viridochromogenes, ssp. komabensis growth may be used in the biochemical synthesis of laspartomycin and selection of such medium is within the capability of those of skill in the art. Representative examples of culturing media which supports Streptomyces viridochromogenes, ssp. komabensis growth may be found in Umezawa et al., U.S. Pat. No. 3,639,582 and Examples 3 and 4.
Preferred media, times, temperatures and pH for culturing Streptomyces viridochromogenes, ssp. komabensis that provide good yields of laspartomycin are described in Umezawa et al., U.S. Pat. No. 3,639,582 and Examples 3 and 4. It should be noted that the choice of culturing medium and the quantitative ratio of its constituents directly affects the ratio of the different lipopeptides that comprise laspartomycin.
Generally, laspartomycin may be purified and isolated by any art-known techniques such as high performance liquid chromatography, counter current extraction, centrifugation, filtration, precipitation, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like. The actual conditions used to purify laspartomycin will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those having skill in the art.
Preferably, laspartomycin is isolated from the culture medium by extractive procedures. In one preferred embodiment (see e.g., Example 5), the fermentation broth containing laspartomycin is mixed with organic solvent (preferably 1-butanol). While not wishing to be bound by theory, the anionic form of laspartomycin may form a chelate with divalent metal ion that is soluble in organic solvent. The organic phase containing laspartomycin is then combined with an aqueous acid solution at a pH less than about 3.0 (most preferably at a pH of about 2.0). While not wishing to be bound by theory, protonation of the anion of laspartomycin may disrupt the divalent metal chelate form.
More preferably, (see e.g., Example 6), the fermentation broth containing laspartomycin is acidified to a pH of at least about 3.0 (more preferably to a pH of about 2.0). The cells and any precipitate may then be separated by any conventional method known to those of skill in the art and suspended in water. The pH of the aqueous suspension is adjusted to at least about pH 7.0, a divalent metal ion is added and the pH of the aqueous suspension is adjusted to about 8.0 to about 9.0. Preferably the concentration of divalent metal ion in the aqueous suspension is between about 4 mmol/l and about 10 mmol/l. In one embodiment, the divalent metal ion is selected from the group consisting of calcium, magnesium and zinc. Most preferably, the divalent metal ion is calcium. The aqueous suspension is then extracted with organic solvent (preferably, 1-butanol). The organic phase containing laspartomycin is then combined with an aqueous acid solution at a pH less than about 3.0 (most preferably at a pH of about 2.0).
Henceforth, in either of the above preferred embodiments, laspartomycin may be partitioned between organic solvent and aqueous solution by conventional methods known to those of skill in the art. Thus, for example, when the organic solvent solution of laspartomycin is treated with a neutral or basic aqueous solution, laspartomycin may be extracted into aqueous solution. Acidification of the aqueous solution of laspartomycin enables extraction of laspartomycin into organic solvent. Preferably, laspartomycin is partitioned between organic solvent and aqueous solution at least twice. Laspartomycin may be isolated as either the free acid (see e.g. Example 7) or a metal salt (see e.g., Examples 5 and 6) using conventional methods known to those of skill in the art.
Generally, the lipophilic moiety of laspartomycin may be cleaved with an enzyme to provide the laspartomycin core peptide. It should be noted that addition of an appropriate enzyme to the culture medium may provide the laspartomycin core peptide directly, thus obviating the need to isolate laspartomycin. Preferably, however, isolated laspartomycin is treated with an enzyme which may be selected by those of skill in the art. The enzyme may be, for example, a degradative enzyme such as a peptidase, esterase or thiolase, of which numerous examples exist in the art. Preferably, the enzyme is a deacylase.
In an exemplary embodiment, the cleavage step involves culturing a microorganism that can produce a deacylase in an appropriate culture medium and contacting laspartomycin with the culture medium containing the deacylase. Microorganisms that produce deacylases are well known to those of skill in the art. In a preferred embodiment, the microorganism Actinoplanes utahensis (NRRL 12052) provides a deacylase.
Parent cultures of Actinoplanes utahensis (NRRL 12052) especially suitable for cleaving the lipophilic fragment of laspartomycin may be selected by methods known to those of skill in the art. A preferred method for selecting a parent culture which provides improved yields of laspartomycin core peptide is described in Example 8.
Growing inocula and inoculating culturing medium are also well known to those of skill in the art and exemplary methods for Actinoplanes utahensis (NRRL 12052) are described in Boeck et al., 1988, J. Antibiot., 41, 1085 and Debono et. al., 1988, J. Antibiotics, 41, 1093 which are herein incorporated by reference and Example 8.
Any culturing medium which supports Actinoplanes utahensis (NRRL 12052) growth may be used and selection of such medium is within the capability of those of skill in the art. Representative examples of culturing medium which supports Actinoplanes utahensis (NRRL 12052) growth may be found in Boeck et al., 1988, J. Antibiot., 41, 1085, Debono et. al., 1988, J. Antibiotics, 41, 1093 and Example 8.
Preferred media, times, temperatures and pH for culturing Actinoplanes utahensis (NRRL 12052) that provide good yields of the deacylase are described in Boeck et al., 1988, J. Antibiot., 41, 1085, Debono et. al., 1988, J. Antibiotics, 41, 1093 and Example 8.
In a preferred embodiment, laspartomycin is contacted with a culture medium containing Actinoplanes utahensis (NRRL 12052) for about 16 hours at about 29xc2x0 C. to provide the laspartomycin core peptide having the structure: 
It should be noted that contacting laspartomycin with a culture medium containing Actinoplanes utahensis (NRRL 12052) for about 4 hours at about 29xc2x0 C. (see e.g., Example 10) provides material enriched in the laspartomycin core peptide having the structure: 
While not wishing to be bound by theory, the deacylase produced by Actinoplanes utahensis (NRRL 12052) may be an exopeptidase that first cleaves the lipophilic fragment of laspartomycin to provide 54. The exocyclic aspartic acid residue of 54 is then hydrolyzed by extended treatment with deacylase or proteases to provide compound 6.
The laspartomycin core peptide may be purified and isolated by any art-known techniques such as high performance liquid chromatography, counter current extraction, centrifugation, filtration, precipitation, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like. The actual conditions used to purify the laspartomycin core peptide will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those having skill in the art. Preferably, the laspartomycin core peptide is isolated by centrifugation and chromatography on reverse phase resin (See e.g., Examples 9 and 10).
4.2.3 Methods of Making Laspartomycin Core Peptide Derivatives
Laspartomycin core peptide derivatives may be made starting from laspartomycin core peptide 6 or laspartomycin core peptide 54. Typically, either 6 or 54 will be produced by deacylation of laspartomycin provided by culturing Streptomyces viridochromogenes, ssp. komabensis (ATCC 29814). However, it may be possible to synthesize either 6 or 54 using methods known in the art for synthesizing cyclic peptides. For example, linear peptides may be prepared using solution phase or solid phase peptide synthesis and then cyclized. Preferably, laspartomycin core peptide 6 will be used as a starting material for the synthesis of laspartomycin core peptide derivatives. Those of skill in the art will realize that any of the methods presented below can also be used to prepare laspartomycin core peptide derivatives from intermediate 54.
Starting materials useful for preparing laspartomycin core peptide derivatives from the laspartomycin core peptide 6 and intermediates thereof are either commercially available or may be prepared by conventional synthetic methods. A number of general synthetic approaches may be envisioned for converting cyclic peptide 6 to laspartomycin core peptide derivatives. These include but are not limited to the approaches outlined in Schemes I-III.
Y1xe2x80x94Lxe2x80x94X1xe2x80x2+NH(R1)xe2x80x94Rxe2x86x92Y1xe2x80x94Lxe2x80x94X1N(R1)xe2x80x94Rxe2x80x83xe2x80x83Scheme 1
In Scheme 1, X1xe2x80x2 may be an activated derivative of X1 such as for example, xe2x80x94COxe2x80x94Z, xe2x80x94OCOxe2x80x94Z, xe2x80x94SO2xe2x80x94Z, xe2x80x94CSxe2x80x94Z, xe2x80x94POxe2x80x94Z, xe2x80x94OPOxe2x80x94Zxe2x80x94, xe2x80x94OC(O)xe2x80x94Z, xe2x80x94NHCOxe2x80x94Z or xe2x80x94NR1COxe2x80x94Z where Z is a leaving group such as halogen or an activated ester. Methods for making activated derivatives of X1 and for reacting these derivatives with either primary or secondary amines to form the X1xe2x80x94N covalent bond are known to those of skill in the art and may be found in any compendium of standard synthetic methods (See e.g., March, J., Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th ed., 1992; Larock, R., Comprehensive Organic Transformations, VCH: New York, 1999; Bodanzsky, M., Principles of Peptide Synthesis; Springer Verlag, 1984; Bodanzsky, M., Practice of Peptide Synthesis; Springer Verlag, 1984). Other synthetic methods based on free radical chemistry, photochemistry or electrochemistry for forming the X1xe2x80x94N bond will be apparent to those of skill in the art.
Those of skill in the art will appreciate that protection of either Y1 and/or L may be necessary to make activated derivatives of X1 for formation of the X1xe2x80x94N bond. In the event that protection of either Y1 and/or L is necessary to form the X1xe2x80x94N linkage, then deprotection of either Y1 and/or L will be necessary to provide the desired laspartomycin core peptide derivative. Methods for protection and deprotection of common organic functionalities are known to those of skill in the art and may be used as necessary in the synthesis of laspartomycin core peptide derivatives (see e.g. Greene, T. W., Protective Groups in Organic Synthesis, 3rd edition, 1999).
Y1xe2x80x94L3+L2xe2x80x94X1xe2x80x94N(R1)Rxe2x86x92Y1xe2x80x94Lxe2x80x94X1N(R1)xe2x80x94Rxe2x80x83xe2x80x83Scheme 2
Scheme 2 describes a convergent approach where Y1xe2x80x94Lxe2x80x94X1N(R1)xe2x80x94R is synthesized by combining two molecules (Y1xe2x80x94L3 and L2xe2x80x94X1xe2x80x94N(R1)R) to form the laspartomycin core peptide derivative. Here L3 and L2 are fragments which, when covalently linked, form the linker L. Such approaches may be particularly useful when L is an oligomer such as a polyamide or poylether. Methods for combining oligomeric subunits such as ether or amide monomers, dimers etc. are known to those of skill in the art. Fragments such as Y1xe2x80x94L3 and L2xe2x80x94X1xe2x80x2 (useful in forming the X1xe2x80x94N bond as described above) are either commercially available or may be made by standard synthetic methods.
Yxxe2x80x94Lxe2x80x94X1N(R1)xe2x80x94Rxe2x86x92Y1xe2x80x94Lxe2x80x94X1N(R1)xe2x80x94Rxe2x80x83xe2x80x83Scheme 3
Finally, simple functional group interchange may be used to prepare Y1xe2x80x94Lxe2x80x94X1N(R1)xe2x80x94R from Yxxe2x80x94Lxe2x80x94X1N(R1)xe2x80x94R. Here, Yx is a functional group that may be converted to Y1. Many methods for effecting functional group interchange are known to those of skill in organic synthesis (See e.g., March, J., Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th ed., 1992; Larock, R., Comprehensive Organic Transformations, VCH: New York, 1999).
4.2.4 The Antimicrobial Laspartomycin Derivatives
The antimicrobial laspartomycin derivatives of the present invention offer some significant advantages over traditional antibiotics. The antimicrobial laspartomycin derivatives are generally active against many gram positive bacteria. More importantly, the antimicrobial laspartomycin derivatives of the present invention may be effective against methicillin resistant bacteria and/or strains resistant to vancomycin. Thus, the antimicrobial laspartomycin derivatives may inhibit or prevent growth of a number of microbes generally resistant to known antibiotics.
Antimicrobial laspartomycin derivatives include compounds described by structural Formula (II):
Y2xe2x80x94(X2xe2x80x94X3)xe2x80x94(L)xe2x80x94(X1)xe2x80x94N(R1)xe2x80x94Rxe2x80x83xe2x80x83(II)
or an pharmaceutically acceptable salt or hydrate thereof, wherein:
Y2 is a lipophilic group;
X2 is a linked group;
X3 is a linked group; and
X1, L, N, R1 and R are as defined for Formula (I) in Section 4.2.1 of this Application.
Connected to X1 in isolated antimicrobial laspartomycin derivatives of Formula (II) is a linking moiety of the formula (X2xe2x80x94X3) where L is a linker and X2 and X3 are linked groups that attach a lipophilic molecule Y2 to the linker L. The nature of linker L and the linked groups X2 and X3 may vary extensively. The linker L has been described and defined in Section 4.2.1 of this Application.
As will be appreciated by those having skill in the art, a linking moiety such as (X2xe2x80x94X3) will typically be at least bifuictional. Thus, they will have at least one functional group or moiety capable of forming a linkage with the linker and at least one functional group or moiety capable of forming a linkage with a lipophilic group.
Preferably, linking moiety (X2xe2x80x94X3) taken together is a covalent linkage. In this preferred embodiment, linking moiety (X2xe2x80x94X3) is any covalent linkage that may be formed by any method known to those of skill in the art. Thus, for example, linking moiety (X2xe2x80x94X3) may be any single, double or triple bond that can be formed between two carbon atoms, a carbon atom and a heteroatom or two heteroatoms. For example, (X2xe2x80x94X3) include linkages such as xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94Cxe2x95x90CHxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94Cxe2x89xa1Cxe2x80x94, xe2x80x94NHxe2x80x94CH2xe2x80x94, xe2x80x94Nxe2x95x90CHxe2x80x94, xe2x80x94CH2xe2x80x94NHxe2x80x94, xe2x80x94CHxe2x95x90Nxe2x80x94, xe2x80x94NHxe2x80x94NHxe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94Sxe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94Oxe2x80x94, xe2x80x94Sexe2x80x94Sexe2x80x94, xe2x80x94Sxe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94Oxe2x80x94, xe2x80x94Sexe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94Sexe2x80x94, xe2x80x94NHxe2x80x94Sxe2x80x94, xe2x80x94Pxe2x80x94Nxe2x80x94, xe2x80x94Nxe2x80x94Oxe2x80x94 and the corresponding substituted analogs where any suitable hydrogen is substituted with the same or different substituent.
Preferably, (X2xe2x80x94X3) taken together are selected from the group consisting of xe2x80x94C(O)Oxe2x80x94, xe2x80x94O(O)Cxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94CONR1xe2x80x94, xe2x80x94NR1COxe2x80x94, xe2x80x94C(O)Sxe2x80x94, xe2x80x94S(O)Cxe2x80x94, xe2x80x94OSO2xe2x80x94, xe2x80x94S(O2)Oxe2x80x94, xe2x80x94NHSO2xe2x80x94, xe2x80x94NR1SO2xe2x80x94, xe2x80x94S(O2)NHxe2x80x94, xe2x80x94S(O2)NR1xe2x80x94, xe2x80x94C(S)NHxe2x80x94, xe2x80x94NHC(S)xe2x80x94, xe2x80x94NHP(O)xe2x80x94, xe2x80x94P(O)NHxe2x80x94, OP(O)xe2x80x94, xe2x80x94P(O)Oxe2x80x94, SP(O)xe2x80x94, xe2x80x94P(O)Sxe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94NHC(O)Oxe2x80x94, xe2x80x94OC(O)NR1xe2x80x94, xe2x80x94NR1C(O)Oxe2x80x94, xe2x80x94OC(O)Oxe2x80x94, xe2x80x94NHC(O)NHxe2x80x94, xe2x80x94NHC(O)NR1xe2x80x94, xe2x80x94NR1C(O)NHxe2x80x94 and xe2x80x94NR1C(O)NR1 and the corresponding substituted analogs where any suitable hydrogen is substituted with the same or different substituent. In a preferred embodiment, (X2xe2x80x94X3) taken together are selected from the group consisting of xe2x80x94C(O)Oxe2x80x94, xe2x80x94O(O)Cxe2x80x94, CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94CONR1xe2x80x94, xe2x80x94NR1COxe2x80x94, xe2x80x94C(O)Sxe2x80x94, xe2x80x94S(O)Cxe2x80x94, xe2x80x94NHSO2xe2x80x94, xe2x80x94NR1SO2, xe2x80x94S(O2)NHxe2x80x94, xe2x80x94S(O2)NR1xe2x80x94, C(S)NHxe2x80x94, xe2x80x94NHC(S)xe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94NHC(O)Oxe2x80x94, xe2x80x94OC(O)NR1xe2x80x94, xe2x80x94NR1C(O)Oxe2x80x94 and xe2x80x94OC(O)Oxe2x80x94 and the corresponding substituted analogs where any suitable hydrogen is substituted with the same or different substituent. In another preferred embodiment, (X2xe2x80x94X3) taken together are selected from the group consisting of xe2x80x94C(O)Oxe2x80x94, xe2x80x94O(O)Cxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94CONR1xe2x80x94, xe2x80x94NR1COxe2x80x94, xe2x80x94NHSO2xe2x80x94, xe2x80x94NR1SO2, xe2x80x94S(O2)NHxe2x80x94, xe2x80x94S(O2)NR1xe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94NHC(O)Oxe2x80x94, xe2x80x94OC(O)NR1xe2x80x94 and xe2x80x94NR1C(O)Oxe2x80x94 and the corresponding substituted analogs where any suitable hydrogen is substituted with the same or different substituent.
Some embodiments of the linking moiety (X2xe2x80x94X3) combined with linker L include partial structures such as xe2x80x94(X2xe2x80x94X3)-(CH2)nxe2x80x94, where n is between 1 and 8, (X2xe2x80x94X3) taken together are selected from the group consisting of xe2x80x94C(O)Oxe2x80x94, xe2x80x94O(O)Cxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94CONR1xe2x80x94, xe2x80x94NR1COxe2x80x94, xe2x80x94NHSO2xe2x80x94, xe2x80x94NR1SO2, xe2x80x94S(O2)NHxe2x80x94, xe2x80x94S(O2)NR1xe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94NHC(O)Oxe2x80x94, xe2x80x94OC(O)NR1xe2x80x94 and xe2x80x94NR1C(O)Oxe2x80x94 and the corresponding analogues where any suitable hydrogen is substituted. Other embodiments of the linking moiety (X2xe2x80x94X3) combined with linker L include representations where X3xe2x80x94L taken together are derived from any amino acid, which may be for example, a D or L xcex1-amino acid, a xcex2-amino acid and a xcex3-amino acid and X2, for example is xe2x80x94COxe2x80x94 or xe2x80x94SO2xe2x80x94. Taken together X3xe2x80x94L also may also be a dipeptide, a tripeptide or a tetrapeptide derivative comprised of any combination of amino acids. The polarity of the peptide bond in these peptides may be either Cxe2x86x92N or Nxe2x86x92C.
Generally, the lipophilic group Y2 will be hydrophobic and when substituted will be substituted with hydrophobic substituents. Those of skill in the art will appreciate that the size and/or length of the lipophilic group will depend, in part, on the nature of fragments such as L, (X2xe2x80x94X3), X1 and R1 that comprise the antimicrobial laspartomycin derivatives.
In a preferred embodiment, the lipophilic group Y2 is selected from the group consisting of (C6-C25) alkyl optionally substituted with one or more of the same or different R2 groups, (C6-C25) heteroalkyl optionally substituted with one or more of the same or different R2 groups, (C8-C25) aryl optionally substituted with one or more of the same or different R2 groups, (C8-C25) arylaryl optionally substituted with one or more of the same or different R2 groups, (C8-C25) biaryl optionally substituted with one or more of the same or different R2 groups, eight to twenty five membered heteroaryl optionally substituted with one or more of the same or different R2 groups, (C8-C25) arylalkyl optionally substituted with one or more of the same or different R2 groups and eight to twenty five membered heteroarylalkyl optionally substituted with one or more of the same or different R2 groups;
each R2 is independently selected from the group consisting of xe2x80x94OR3, xe2x80x94SR3, xe2x80x94NR3R3, xe2x80x94CN, xe2x80x94NO2, xe2x80x94N3, xe2x80x94C(O)OR3, xe2x80x94C(O)NR3R3, xe2x80x94C(S)NR3R3, xe2x80x94C(NR3)NR3R3, xe2x80x94CHO, xe2x80x94R3CO, xe2x80x94SO2R3, xe2x80x94SOR3, xe2x80x94PO(OR3)2, xe2x80x94PO(OR3), xe2x80x94CO2H, xe2x80x94SO3H, xe2x80x94PO3H, halogen and trihalomethyl;
each R3 is independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C5-C10) aryl, 5-10 membered heteroaryl, (C6-C16) arylalkyl and 6-16 membered heteroarylalkyl.
In a more preferred embodiment, the lipophilic group Y2 is selected from the group consisting of (C8-C20) alkyl optionally substituted with one or more of the same or different R2 groups, (C8-C20) heteroalkyl optionally substituted with one or more of the same or different R2 groups, (C8-C20) aryl optionally substituted with one or more of the same or different R2 groups, (C8-C20) arylaryl optionally substituted with one or more of the same or different R2 groups, (C8-C20) biaryl optionally substituted with one or more of the same or different R2 groups, eight to twenty membered heteroaryl optionally substituted with one or more of the same or different R2 groups, (C8-C20) arylalkyl optionally substituted with one or more of the same or different R2 groups and eight to twenty membered heteroarylalkyl optionally substituted with one or more of the same or different R2 groups where R2 is as defined above.
In one preferred embodiment, the lipophilic group Y2 is selected from the group consisting of (C8-C20) alkyl optionally substituted with one or more of the same or different R2 groups, (C8-C20) heteroalkyl optionally substituted with one or more of the same or different R2 groups, (C8-C20) aryl optionally substituted with one or more of the same or different R2 groups, (C8-C20) arylaryl optionally substituted with one or more of the same or different R2 groups, (C8-C20) biaryl optionally substituted with one or more of the same or different R2 groups, ten to twenty membered heteroaryl optionally substituted with one or more of the same or different R2 groups, (C8-C20) arylalkyl optionally substituted with one or more of the same or different R2 groups and ten to twenty membered heteroarylalkyl optionally substituted with one or more of the same or different R2 groups. In another preferable embodiment, the lipophilic group Y2 is selected from the group consisting of (C8-C20) alkyl optionally substituted with one or more of the same or different R2 groups. In yet another preferable embodiment, the lipophilic group Y2 is selected from the group consisting of (C10-C16) alkyl optionally substituted with one or more of the same or different R2 groups.
In an exemplary embodiment of the isolated antimicrobial laspartomycin derivative of Formula (II), X1 is xe2x80x94COxe2x80x94 or xe2x80x94SO2xe2x80x94, (X2xe2x80x94X3) taken together are selected from the group consisting of xe2x80x94C(O)Oxe2x80x94, xe2x80x94O(O)Cxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94C(O)Sxe2x80x94, xe2x80x94S(O)Cxe2x80x94, xe2x80x94OSO2xe2x80x94, xe2x80x94S(O2)Oxe2x80x94, xe2x80x94NHSO2xe2x80x94, xe2x80x94S(O2)NHxe2x80x94, xe2x80x94C(S)NHxe2x80x94, xe2x80x94NHC(S)xe2x80x94, xe2x80x94NHP(O)xe2x80x94, xe2x80x94P(O)NHxe2x80x94, OP(O)xe2x80x94, xe2x80x94P(O)Oxe2x80x94, xe2x80x94SP(O)xe2x80x94, xe2x80x94P(O)Sxe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94NHC(O)Oxe2x80x94, xe2x80x94OC(O)NR1xe2x80x94, xe2x80x94NR1C(O)Oxe2x80x94, xe2x80x94OC(O)Oxe2x80x94, xe2x80x94NHC(O)NHxe2x80x94, xe2x80x94NHC(O)NR1xe2x80x94 and xe2x80x94NR1C(O)Oxe2x80x94, R1 is hydrogen and L is selected from the group consisting of L1, L2, L3 and L4 where L1, L2, L3 and L4 are as defined in Section 4.2.1 of this Application
In a preferred embodiment, S1 is a side chain of a genetically encoded xcex1 amino acid. Exemplary preferred embodiments of Y2xe2x80x94(X2xe2x80x94X3)xe2x80x94Lxe2x80x94X1xe2x80x94N(R1)xe2x80x94R where K is independently selected from the group consisting of oxygen, nitrogen and sulfur include the following compounds where Y2, X2, X3 and R are as previously defined: 
Preferably, in the these embodiments, X3 is selected from the group consisting of xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94NHxe2x80x94 and X2 is selected from the group consisting of xe2x80x94COxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94NHC(O)xe2x80x94 and xe2x80x94NR1C(O)xe2x80x94. In an alternative embodiment, X2 is selected from the group consisting of xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94NHxe2x80x94 and X3 is selected from the group consisting of xe2x80x94COxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94NHC(O)xe2x80x94 and xe2x80x94NR1C(O)xe2x80x94.
In another preferred embodiment of the antimicrobial laspartomycin derivatives, X1 is xe2x80x94COxe2x80x94 or xe2x80x94SO2xe2x80x94, (X2xe2x80x94X3) taken together are selected from the group consisting of xe2x80x94CONHxe2x80x94, xe2x80x94S(O2)NHxe2x80x94, xe2x80x94C(S)NHxe2x80x94, xe2x80x94P(O)NHxe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94OC(O)NR1xe2x80x94, xe2x80x94NHC(O)NHxe2x80x94, and xe2x80x94NHC(O)NR1, R1 is hydrogen, n is as defined in Section 4.2.1 of this Application and L is L1 as defined in Section 4.2.1 of this Application. Preferably, in this embodiment, each S1 is independently a side-chain of a genetically encoded xcex1-amino acid. More preferably, each S1 is independently a side-chain of glycine asparagine, aspartic acid, glutamine, glutamic acid, tryptophan, phenylalanine, tyrosine, leucine, alanine, isoluecine and valine. Exemplary preferred embodiments of Y2xe2x80x94(X2xe2x80x94X3)xe2x80x94Lxe2x80x94X1xe2x80x94N(R1)xe2x80x94R where each S1 is independently a side-chain of glycine asparagine, aspartic acid, glutamine, glutamic acid and tryptophan include the following compounds where Y2, (X2xe2x80x94X3) taken together and R are as previously defined: 
Preferably, in the these embodiments X3 is selected from the group consisting of xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94NHxe2x80x94 and X2 is selected from the group consisting of xe2x80x94COxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94NHC(O)xe2x80x94 and xe2x80x94NR1C(O)xe2x80x94. In alternative embodiment, X2 is selected from the group consisting of xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94NHxe2x80x94 and X3 is selected from the group consisting of xe2x80x94COxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94NHC(O)xe2x80x94 and xe2x80x94NR1C(O)xe2x80x94. Preferably, in the above depicted embodiments the illustrated a amino acids have the L stereochemistry.
In a preferred embodiment X2xe2x80x94X3 taken together are xe2x80x94CONHxe2x80x94 or xe2x80x94SO2NHxe2x80x94. Most preferably, X2xe2x80x94X3 taken together are xe2x80x94CONHxe2x80x94. Particularly preferred embodiments of Y2 include tetradecan-1-yl, nonan-1-yl, decan-1-yl and 12-methyl-tridecan-1-yl.
Exemplary preferred isolated antimicrobial laspartomycin derivatives according to structural formula II include: 
Preferably, in the above depicted embodiments, the polyamide linkers depicted have the L stereochemistry at the xcex1 carbon of the illustrated amino acids.
4.2.5 Methods of Making the Antimicrobial Laspartomycin Derivatives
Antimicrobial laspartomycin derivatives may be synthesized from laspartomycin core peptide 6, laspartomycin core peptide 54 and laspartomycin core peptide derivatives of Formula (I). Laspartomycin core peptide derivatives of Formula (I) may be synthesized by the approaches outlined in Section 4.2.2 of this Application. Those of skill in the art will appreciate that other starting materials may be used in the synthesis of antimicrobial laspartomycin derivatives.
A number of general synthetic approaches may be envisioned for converting laspartomycin core peptide 6, laspartomycin core peptide 54 and laspartomycin core peptide derivatives of Formula I to antimicrobial laspartomycin derivatives. These include but are not limited to the approaches outlined in Schemes 4 and 5.
Y2xe2x80x94X2xe2x80x94X3xe2x80x94Lxe2x80x94X1xe2x80x2+HN(R1)xe2x80x94Rxe2x86x92Y2xe2x80x94X2xe2x80x94X3xe2x80x94Lxe2x80x94X1N(R1)xe2x80x94Rxe2x80x83xe2x80x83Scheme 4
In Scheme 4 a lipophilic fragment Y2 and a linker L, attached via linked groups X2 and X3 are covalently linked to X1xe2x80x2 which may be an activated derivative of X1 such as for example, xe2x80x94COxe2x80x94Z, xe2x80x94OCOxe2x80x94Z, xe2x80x94SO2xe2x80x94Z, xe2x80x94CSxe2x80x94Z, xe2x80x94POxe2x80x94Z, xe2x80x94OPOxe2x80x94Z, xe2x80x94OC(O)xe2x80x94Z, xe2x80x94NHCOxe2x80x94Z or xe2x80x94NR1COxe2x80x94Z where Z is a leaving group such as halogen or an activated ester. Methods for making activated derivatives of X1 and for reacting these derivatives with either primary or secondary amines to form the X1xe2x80x94N covalent bond are known to those of skill in the art and may be found in any compendium of standard synthetic methods (See e.g., March, J., Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th ed., 1992; Larock, R., Comprehensive Organic Transformations, VCH: New York, 1999; Bodanzsky, M., Principles of Peptide Synthesis; Springer Verlag, 1984; Bodanzsky, M., Practice of Peptide Synthesis; Springer Verlag, 1984). Other synthetic methods based on free radical chemistry, photochemistry or electrochemistry for forming the X1xe2x80x94N bond will be apparent to those of skill in the art. Formation of the X1xe2x80x94N covalent bond provides the antimicrobial laspartomycin derivative. Methods for making (X2xe2x80x94X3) linkages such as esters, amides phosphoramidites, sulfonamides, carbamates, ureas etc. are also conventional and known to those of skill in the art (See e.g., March, J., Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th ed., 1992; Larock, R., Comprehensive Organic Transformations; VCH: New York, 1999; Bodanzsky, M., Principles of Peptide Synthesis; Springer Verlag, 1984; Bodanzsky, M., Practice of Peptide Synthesis; Springer Verlag, 1984)
xe2x80x83Y2xe2x80x94X2xe2x80x2+Y1xe2x80x94Lxe2x80x94X1N(R1)xe2x80x94Rxe2x86x92Y2xe2x80x94X2xe2x80x94X3xe2x80x94Lxe2x80x94X1N(R1)xe2x80x94Rxe2x80x83xe2x80x83Scheme 5
Scheme 5 describes a convergent approach where Y2xe2x80x94X2xe2x80x2 (X2xe2x80x2 is a derivative of the linked group X2) and Y1xe2x80x94Lxe2x80x94X1N(R1)xe2x80x94R are combined to form the (X2xe2x80x94X3) linkage thus providing the antimicrobial laspartomycin derivative. Methods for forming the (X2xe2x80x94X3) linkage are described above. Fragments such as Y2xe2x80x94X2xe2x80x2 are either commercially available or may be made by standard synthetic methods. Y1xe2x80x94Lxe2x80x94X1N(R1)xe2x80x94R may be made as described in Section 4.2.2 of this application.
Those of skill in the art will appreciate that protection of either Y2 and/or L may be necessary to form (X2xe2x80x94X3) linkage. In the event that protection of either Y2 and/or L is necessary to form the (X2xe2x80x94X3) linkage, then deprotection of either Y2 and/or L will be necessary to provide the antimicrobial laspartomycin derivative. Methods for protection and deprotection of common organic functionalities are known to those of skill in the art and may be used as necessary in the synthesis of antimicrobial laspartomycin derivatives (see e.g. Greene, T. W., Protective Groups in Organic Synthesis, 3rd edition, 1999).
4.2.6 Methods of Inhibiting Microbial Growth
Generally, active isolated antimicrobial laspartomycin derivatives of the invention are identified using in vitro screening assay. Indeed, in many instances the isolated antimicrobial laspartomycin derivatives of the invention will be used in vitro as preservatives, topical antimicrobial treatments, etc. Additionally, despite certain apparent limitations of in vitro susceptibility tests, clinical data indicate that a good correlation exists between minimal inhibitory concentration (MIC) test results and in vivo efficacy of antibiotic compounds (Murray, 1994, Antimicrobial Susceptibility Testing, Poupard et al., eds., Plenum Press, N.Y.; Knudsen et al., 1995, Antimicrob. Agents Chemother. 39 (6):1253-1258). Thus, isolated antimicrobial laspartomycin derivatives useful for treating infections and diseases related thereto are also conveniently identified by demonstrated in vitro antimicrobial activity against specified microbial targets.
Generally, the in vitro antimicrobial activity of antimicrobial agents is tested using standard NCCLS bacterial inhibition assays, or MIC tests (see, National Committee on Clinical Laboratory Standards xe2x80x9cPerformance Standards for Antimicrobial Susceptibility Testing,xe2x80x9d NCCLS Document M100-S5 Vol. 14, No. 16, December 1994; xe2x80x9cMethods for dilution antimicrobial susceptibility test for bacteria that grow aerobically-Third Edition,xe2x80x9d Approved Standard M7-A3, National Committee for Clinical Standards, Villanova, Pa.).
Alternatively, the antimicrobial laspartomycin derivatives of the invention may be assessed for antimicrobial activity using in vivo models. Again, such models are well-known in the art.
It will be appreciated that other assays, that are well known in the art or which will become apparent to those having skill in the art upon review of this disclosure, may also be used to identify active isolated antimicrobial laspartomycin derivatives of the invention. Such assays include, for example, the assay described in Lehrer et al., 1988, J. Immunol. Methods 108:153 and Steinberg and Lehrer, xe2x80x9cDesigner Assays for Antimicrobial Peptides: Disputing the xe2x80x98One Size Fits Allxe2x80x99 Theory,xe2x80x9d In: Antibacterial Peptide Protocols, Shafer, Ed., Humana Press, N.J.
Generally, isolated antimicrobial laspartomycin derivatives of the invention will exhibit MICs of less than about 64 xcexcg/mL, usually less than about 32 xcexcg/mL, preferably less than about 16 xcexcg/mL and most preferably less than about 4 xcexcg/mL. The antimicrobial laspartomycin derivatives of the invention may also exhibit antifungal activity, having MICs of about 50 xcexcg/mL or less against a variety of fungi in standard in vitro assays.
Of course, compounds having MICs on the low end of these ranges, or even lower, are preferred. Most preferred for use in treating or preventing systemic infections are antimicrobial laspartomycin derivatives that exhibit significant antimicrobial activity (i.e., less than 4 xcexcg/mL), good water-solubility (at approx. neutral pH) and low toxicity. Toxicity is less of a concern for topical administration, as is water solubility.
4.2.7 Other Methods and Pharmaceutical Compositions
The antimicrobial laspartomycin derivatives of the invention can be used in a wide variety of applications to inhibit the growth of microorganisms or kill microorganisms. For example, the antimicrobial laspartomycin derivatives may be used as disinfectants or as preservatives for materials such as foodstuffs, cosmetics, medicaments and other nutrient containing materials. The antimicrobial laspartomycin derivatives can also be used to treat or prevent diseases related to microbial infection in subjects such as plants and animals.
For use as a disinfectant or preservative, the antimicrobial laspartomycin derivatives can be added to the desired material singly, as mixtures of antimicrobial laspartomycin derivatives, or in combination with other antifungal and/or antimicrobial agents. The antimicrobial laspartomycin derivatives may be supplied as the compound per se or may be in admixture with a variety of carriers, diluents or excipients, which are well known in the art.
When used to treat or prevent microbial infections or diseases related thereto the antimicrobial laspartomycin derivatives of the invention can be administered or applied singly, as mixtures of two or more antimicrobial laspartomycin derivatives, in combination with other antifungal, antibiotic or antimicrobial agents or in combination with other pharmaceutically active agents. The antimicrobial laspartomycin derivatives can be administered or applied per se or as pharmaceutical compositions. The specific pharmaceutical formulation will depend upon the desired mode of administration, and will be apparent to those having skill in the art. Numerous compositions for the topical or systemic administration of antibiotics are described in the literature. Any of these compositions may be formulated with the antimicrobial laspartomycin derivatives of the invention.
Pharmaceutical compositions comprising the antimicrobial laspartomycin derivatives of the invention may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the active antimicrobial laspartomycin derivatives into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For topical administration the antimicrobial laspartomycin derivatives of the invention may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.
Systemic formulations include those designed for administration by injection, e.g. subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.
For injection, the antimicrobial laspartomycin derivatives of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks""s solution, Ringer""s solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the antimicrobial laspartomycin derivatives may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the antimicrobial laspartomycin derivatives can be readily formulated by combining them with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms may be sugar-coated or enteric-coated using standard techniques.
For oral liquid preparations such as, for example, suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like may be added.
For buccal administration, the compositions may take the form of tablets, lozenges, etc. formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The antimicrobial laspartomycin derivatives may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the antimicrobial laspartomycin derivatives may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well known examples of delivery vehicles that may be used to deliver the antimicrobial laspartomycin derivatives of the invention. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the antimicrobial laspartomycin derivatives may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
As certain of the carboxylic acids of the antimicrobial laspartomycin derivatives of the invention are acidic, or the lipophilic group or linker may include acidic or basic substituents, the antimicrobial laspartomycin derivatives may be included in any of the above-described formulations as the free acids, the free bases or as pharmaceutically acceptable salts. Pharmaceutically acceptable salts are those salts which retain substantially the antimicrobial activity of the free acids or bases and which are prepared by reaction with bases or acids, respectively. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base or acid forms.
The antimicrobial laspartomycin derivatives of the invention, or compositions thereof, will generally be used in an amount effective to achieve the intended purpose. Of course, it is to be understood that the amount used will depend on the particular application.
For example, for use as a disinfectant or preservative, an antimicrobially effective amount of a antimicrobial laspartomycin derivative, or composition thereof, is applied or added to the material to be disinfected or preserved. By antimicrobial effective amount is meant an amount of antimicrobial laspartomycin derivative or composition that inhibits the growth of, or is lethal to, a target microbe. While the actual amount will depend on a particular target microbe and application, for use as a disinfectant or preservative the antimicrobial laspartomycin derivatives, or compositions thereof, are usually added or applied to the material to be disinfected or preserved in relatively low amounts. Typically, the antimicrobial laspartomycin derivatives comprises less than about 5% by weight of the disinfectant solution or material to be preserved, preferably less than about 1% by weight and more preferably less than about 0.1% by weight. An ordinarily skilled artisan will be able to determine antimicrobially effective amounts of particular antimicrobial laspartomycin derivatives for particular applications without undue experimentation using, for example, the in vitro assays provided in the examples.
For use to treat or prevent microbial infections, the antimicrobial laspartomycin derivatives of the invention, or compositions thereof, are administered or applied in a therapeutically effective amount. By therapeutically effective amount is meant an amount effective to ameliorate the symptoms of, or ameliorate, treat or prevent microbial infections. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
As in the case of disinfectants and preservatives a therapeutically effective dose, for topical administration to treat or prevent microbial, yeast, fungal or other infection, can be determined using, for example, the in vitro assays provided in the examples. The treatment may be applied while the infection is visible, or even when it is not visible. An ordinarily skilled artisan will be able to determine therapeutically effective amounts to treat topical infections without undue experimentation.
For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating antimicrobial laspartomycin derivative concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of test compound that is lethal to 50% of a cell culture), the MIC as determined in cell culture (i.e., the minimal inhibitory concentration for growth) or the IC100 as determined in cell culture (i.e., the concentration of antimicrobial laspartomycin derivative that is lethal to 100% of a cell culture). Such information can be used to more accurately determine useful doses in humans.
Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art can readily optimize administration to humans based on animal data.
Alternatively, initial dosages can be determined from the dosages administered of known antimicrobial agents (e.g., laspartomycin) by comparing the IC50, MIC and/or I100 of the specific antimicrobial laspartomycin derivatives with that of a known antimicrobial agent, and adjusting the initial dosages accordingly. The optimal dosage may be obtained from these initial values by routine optimization.
Dosage amount and interval may be adjusted individually to provide plasma levels of the active antimicrobial laspartomycin derivatives which are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5 to 1 mg/kg/day. Therapeutically effective serum levels may be achieved by administering a single daily dose or multiple doses each day.
In cases of local administration or selective uptake, the effective local concentration of antimicrobial laspartomycin derivative may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
The amount of antimicrobial laspartomycin derivative administered will, of course, be dependent on, among other factors, the subject being treated, the subject""s weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
The antimicrobial therapy may be repeated intermittently while infections are detectable, or even when they are not detectable. The therapy may be provided alone or in combination with other drugs, such as for example other antibiotics or antimicrobials, or other antimicrobial laspartomycin derivatives of the invention.
Preferably, a therapeutically effective dose of the antimicrobial laspartomycin derivatives described herein will provide therapeutic benefit without causing substantial toxicity. Toxicity of the antimicrobial laspartomycin derivatives can be determined using standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. Antimicrobial laspartomycin derivatives which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in subjects. The dosage of the antimicrobial laspartomycin derivatives described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient""s condition (See, e.g. Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.1, p.1).